Process for the simultaneous determination of glucose and fructose

ABSTRACT

PROCESS FOR THE SIMULTANEOUS DETERMINATION OF GLUCOSE AND FRUCTOSE IN AQUEOUS SOLUTIONS THEREOF, HAVING A TEMPERATURE BETWEEN 50 AND 75*C., SUCH AS THE WARM ELUATES RECOVERED FROM AN ION EXCHANGE APPARATUS USED TO SEPARATE AN INVERT SUGAR SOLUTION INTO ITS COMPONENTS, WHICH INVOLVES MEASURING THE OPTICAL ROTATION AND REFRACTIVE INDEX OF THE WARM SOLUTIONS AND RELATING THE MEASUREMENTS TO THE GLUCOSE AND FRUCTOSE CONCENTRATIONS IN THE SOLUTIONS.

Sept. 26, 1972 K, uER Er AL 3,594,158

PROCESS FOR THE SIMULTANEOUS DETERMINATION OF GLUCOSE AND FRUC'IOSE Original Filed Aug. 1. 1966 3 Sheets-Sheet 1 FIG. I.

60 0C 0 Sf IN THE ZCM.TUBE

200 I: f 0" i- 3 1,3700 5 P 13600 |.3500 5- |.34o0 j '00 E |.330o 5- INVENTORS KARL LAUER HELMUT SPINGLER KARL-ERHARD WALLACH BY GEORG ST ECK W ZLAT RN Sept. 26, 1972 K. LAUER ETAL 3,694,158

PROCESS FOR THE SIMULTANEOUS DETERMINATION OF GLUCOSE AND FRUCTOSE Original Filed Aug. 1, 1966 3 Sheets-Sheet 2 FIG. 2.

I50 A I a U1 GRAMS PER LITER 0 30 6O 90 :20 I50 I80 m [\VEYTORS l6 LITERS PER HOUR KAF-eL L-AUER HELMUT SPINGLER B Y KARL-ERHARD WALLACF GEORG ST ECK Sept. 26, 1972 K. LAUER ETAL 3,694,158

PROCESS FOR THE SIMULTANEOUS DETERMINATION OF GLUCOSE AND FRUCTOSE Original Filed Aug. 1, 1966 3 Sheets-Sheet 3 FIG. 3.

GRAMS PER LITER5 (J1 Mi ,wmoRs w KARL LAUER HELMUT SDINGLER BY KARL-ERHARO WALLACH GEORG STO K @444/ o,

United States Patent Oifi ce Patented Sept. 26, 1972 84, 8 Int. Cl. G01n 21/40, 21/46 US. Cl. 23-230 R 7 Claims ABSTRACT OF THE DISCLOSURE Process for the simultaneous determination of glucose and fructose in aqueous solutions thereof, having a temperature between 50 and 75 C., such as the warm eluates recovered from an ion exchange apparatus used to separate an invert sugar solution into its components, which involves measuring the optical rotation and refractive index of the warm solutions and relating the measurements to the glucose and fructose concentrations in the solutions.

The present invention is concerned with a process for the simultaneous determination of glucose and fructose, and more particularly with the simultaneous determination, without delay, in warm (50 to 75 C.), aqueous solutions.

The process of the type provided by the present invention is an essential requirement for being able to carry out, on a large scale, the separation of glucose and fructose on ion exchanger columns (c.f. US. Pat. No. 3,044,904 and US. patent application Ser. No. 569,362, filed Aug. 1, 1966, now abandoned. All previously used and known analytical procedures for the determination of glucose and fructose cannot be used for the abovementioned purpose because the determination of the two components in a continuously operating process is too complicated and, in particular, is too laborious.

It is, therefore, an object of the invention to provide a simple, quick and continuous determination of glucose and fructose in eluates derived from ion exchange columns, in which an invert sugar solution has been separated into its components.

Another object of this invention is a process for the simultaneous determination of glucose and fructose in eluates derived from ion exchange columns, wherein the analytical data can be achieved in the form of proportional voltages.

A further object of this invention is a process for the determination of glucose and fructose which can be used for the automatic control of the flow of the eluates of ion exchange columns.

These and further objects of this invention will become apparent from a study of the within specification and accompanying examples.

In accordance with the invention, it has now been found that glucose and fructose can be determined simultaneously from warm aqueous solutions thereof when the angle of rotation (at) and the refractive index (n) of these solutions are measured and the concentrations of the components calculated therefrom in known manner. The applicants herein have surprisingly found that this method of analysis can be relied on although the final optical rotation of glucose-fructose solutions is, upon cooling, only slowly adjusted (i.e. in 0.5-1 hour) (c.f.

also Browne and Zerban, Sugar Analysis, 3rd edition, publ. John Wiley & Sons Inc., 1941, page 293). A rapid measurement of the glucose/fructose content of warm, aqueous solutions, after cooling to the conventional measurement temperature of 20 C., as heretofore employed, is not possible.

Surprisingly, it has also been found that, in spite of the above-mentioned difficulties, glucose and fructose can be determined simultaneously and substantially without delay by means of the refractive index and angle of rotation when the measurement of these two physical proper ties is not carried out, as is usual, at 20 C. but at a temperature which is the same as or higher than the temperature of the original solution. The adjustment of the optical rotation then takes place so quickly that the values obtained can even be used, via an automatic computer, for example, an analogue computer, for the automatic control of the separating column.

This result was not to have been foreseen and depends upon a previously unknown phenomenon for which hitherto no explanation has been found. Thus, if an attempt is made to determine the temperature dependence of the optical rotation of fructose, then, for each measurement point, a waiting period of more than one hour is necessary until a sufficiently constant optical rotation is obtained, with the understanding that the fructose solutions in question have been cooled from a higher temperature to the measurement temperature. If, on the other hand, the fructose solution is warmed up from a lower temperature to the measurement temperature, then, surprisingly, the optical rotation is adjusted in practically a matter of seconds. By means of this discovery, it was possible, for the first time, simultaneously to determine glucose and fructose in warm, aqueous solutions by measurement of the angle of rotation (at) and of the refractive index (n) and thus to arrive at a technically extraordinarily important process.

It was not obvious to carry out the simultaneous determination of glucose and fructose as herein disclosed for the reasons that:

(a) the above-mentioned effect of the adjustment of the optical rotation of fructose was unknown, and

(b) it is naturally wearisome work (instead of the values of 20 C. available from the literature) to determine new calibration values for higher temperatures, to determine functional equations from the calibration values and to calculate nomograms therefrom.

For the carrying out of the process according to the present invention, in one part of a warm solution emerging from the end of an ion exchanger column, which contains varying amounts of glucose and fructose, there are meassured the angle of rotation and refractive index at the same or somewhat higher temperature.

As measuring devices for the angle of rotation and refractive index, there can, in principle, be used all commercially available polarimeters and refractometers with flow-through cuvettes. The values so obtained are evaluated in the usual manner by means of a nomogram. The nomograms are calculated by series of calibration values and the functional equations determined thereon. Nomograms allow easy and quick evaluations even by untrained people. However, it is preferable to use continuously registering, well thermostated polarimeters and refractographs in which the solutions to be measured, after passing therethrough, can be returned to the liquid emerging from the separating column. In general, these continuously operating devices also permit the measurement results to be taken off simultaneously as proportional voltages which can be fed into an automatic computer, preferably a programcontrolled computer, the results of which can be used for controlling the whole plant, or can be supplied to multicolor recording aparatus, the results then being obtained in the form of a diagram.

In the accompanying drawings,

FIG. 1 shows such a new nomogram for evaluating the amounts of fructose and glucose in a 60 C. warm aqueous solution by means of the angle of rotation and the refractive index at 60 C. being achieved by a series of calibration values at 60 C.;

FIG. 2 shows a diagram which was prepared from a series of single evaluations according to this invention by means of the nomogram, of the sugar content of the effluent of an ion exchange resin column separating fructose and glucose at 60 C., and

FIG. 3 shows a diagram which was prepared from an analogous series of single evaluations as in FIG. 2, the measurement however being done at 20 C. by prior cooling of the eflluent to 20 C. and using a 20 C. nomogram. The fructose seems still to contain substantially high amounts of glucose, though as can be shown by checking this result by means of conventional analysis, the fructose fractions contain less or no glucose.

The process according to the present invention is, of course, not only suitable for the analysis of eluates of invert sugar solutions separated on ion exchangers (for example according to US. Pat. No. 3,044,904 and copending US. application Ser. No. 569,362 according to German patent application No. B 83,146 IVa/89i), but is, in principle, suitable for the analysis of all warm sugar solutions on a technically satisfactory scale.

The following examples are given for the purpose of illustrating the present invention; however, they are not intended to limit the scope of this invention:

EXAMPLE 1 Discontinuous, rapid determination of glucose and froctose in eluates from an ion exchanger column An invert sugar solution with a temperature of 60 C. emerging from the lower end of a calcium ion-charged cationic exchanger column, which has been more or less separated into glucose and fructose (c.f.,, US. Pat. No. 3,044,904 and copending US. application Ser. No. 569,362 corresponding to German patent application No. B 83,146 IV-a/89i) was, in part, passed via a shortest possible polyethylene tube, first, through a polarimeter (Perkin Elmer) and, secondly, through a refractometer (Zeiss), both of which are provided with flow-through cuvettes, and subsequently again returned to the main current of liquid. The values were read off every minutes and evaluated in the usual manner by means of a nomogram calculated for 60 C.

The nomogram (see FIG. 1 of the accompanying drawings) was calulated from the corresponding calibration curves. The concentration values for glucose and fructose were incorporated into a graph (see FIG. 2 of the accompanying drawings) and showed that glucose and fructose emerged from the column substantially separated. Fractions which preponderantly contained one of the components were collected separately and worked up to give fructose or glucose. Fractions which still contained considerable amounts of both components were combined with the original invert sugar solution and again subjected to a chromatographic separation.

EXAMPLE 2 For comparison, the sugar solution emerging from the column, before entering into the measuring devices, was passed through a cooler and thus cooled to exactly 20 C. The refractive index and optical rotation were read oif every 10 minutes and evaluated by means of a nomogram calculated for 20 C. The values for glucose and fructose were, in the manner described above, incorporated into a graph (see FIG. 3 of the accompanying drawings); this showed that the fructose was apparently still contaminated with considerable amounts of glucose. The re-examination of this result with previously used but time-consuming analytical procedures for the determination of fructose and glucose showed that false results had clearly been obtained which were due to the slow adjsutment to the final optical rotation of the fructose.

EXAMPLE 3 Measuring at higher temperatures than the temperatures of the eflluent of a chromatographic column The part of the sugar solution withdrawn from the main current of the eluate from a chromatographic column, as in Example 1, but which was operated at 55 C., was warmed in a thermostatically controlled water bath to exactly 60 C. before measurement. The values for glucose and fructose, determined by means of a nomogram from the values for the refractive index and the optical rotation, gave an elution graph similar to FIG. 1.

EXAMPLE 4 Continuous, rapid determination of glucose and fructose in eluates from an ion exchanger column A part of the eluate emerging at a temperature of 60 C. from the end of an ion exchanger column was passed, via the shortest possible polyethylene tube, through a continuously registering polarimeter (Zeiss) and through a continuously registering refractograph (Zeiss), from which the measurement values could also be taken off in the form of a proportional voltage. The current of liquid was subsequently again returned to the main current of the eluate. The measurement voltages of the polarimeter and of the refractograph were fed into an analogue computor (Zeiss) which gave the concentrations of glucose and fructose, with the help of a multi-color recording device, as a continuous elution diagram similar to that in FIG. 2.

The following, empirically determined functional equations, which are valid for a temperature of 60 C., were applied to the analogue computer:

c=c +c =total concentration (g./ liter) n=refractive index [oz :specific rotation for glucose [a =specific rotation for fructose The concentrations for glucose and fructose were obtained from the following equations:

1000 awn-0+ c G [whi h Fa l cki r] wherein c .=glucose concentration (g./liter) c =fructose concentration (g./liter) x=length of the polarimeter tube in dm. a=angle of rotation in degrees The measurement of the optical rotation (at) at 546 mm. (Hg line), instead of at the usual Na line, was made because of the longer life and the higher light intensity of mercury lamps.

By the provision of a control program in the analogue computer, valves were operated which directed into three separate containers the fractions which follow one another, i.e. glucose, glucose+fructose, fructose, fructose-l-glucose, glucose, etc. Upon the exceeding of the permitted limiting concentration of one of the components in the other, the solutions in question were passed into a collecting vessel for mixed fractions which were mixed with fresh invert sugar and subsequently again passed through the separating column. The fractions consisting of pure fructose solution and pure glucose solution were worked up separately in the conventional manner, for example by evaporation and crystallization.

What is claimed is:

1. Process for the continuous and simultaneous determination of glucose and fructose in an aqueous solution thereof having a temperature of about 50 to about 75 C., comprising measuring the optical rotation and refractive index of said solution by means of a continuously registering polarimeter and refractograph, and relating said measurements to the concentrations of both glucose and fructose in said solution.

2. Process for the continuous and simultaneous determination of glucose and fructose in an aqueous solution thereof having a temperature of about 50 to about 75 0., comprising measuring of the optical rotation and refractive index of said solution by means of a continuously registering polarimeter and refractograph, using the optical rotation and refractive index measurements, respectively, to calculate therefrom the concentrations of glucose and fructose, respectively in said solution by comparison of such measurements with measurements of a known sample or known samples.

3. Process according to claim 2 wherein said aqueous solution of glucose and fructose is the eluate from an ion exchanger in which an invert sugar solution has been separated into its components.

4. Process according to claim 2 wherein the measurements are evaluated by means of a nomogram based on 'a series of calibration values, to yield the glucose and fructose concentrations in said solution.

5. Process according to claim 2 in which said measurements taken by means of a continuously registering polarimeter and refractograph are taken off in the form of proportional signals.

6. Process according to claim 5 which comprises utilizing said signals in a data processing system which in turn is adapted to control the manufacture of said glucose and fructose in aqueous solution.

7. Process according to claim 5 in which said signals are in the form of proportional voltages.

References Cited UNITED STATES PATENTS 7/1962 Serbia 127-46 OTHER REFERENCES MORRIS O. WOLK, Primary Examiner S. MARANTZ, Assistant Examiner US. Cl. X.R.

UNITED s'iA'wcs PA'ITICN'I. OFFICE CERTEFICATE OF CORKMI'IIUN patent NO, 3,694,158 Dated September 26, 1972 Invcntor(s) Karl Lauer at a].

It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Col. 1 head,

the name of the assignee should reed 4 Boehringer Mannheim GMBHL,

Mannheim, Germany Col. 3 line 35 For "froctose" Read Y fructose -1 4, n For "adjsutment" d i adjustment Col. 4, line 40,

Read h e: 1.38

ORM Po-iOSO (10-69) USCOMM-DC 60376-P69 patent 3,694,158 D d September 26 1972 Inventoflg) Karl Lauer at a].

It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

Col. 6, line 22,

For "22-31" Read e 222-31 Signed ahd sealed this 10th day of April 1973.

(SEAL) Attest:

EDWARD M.FLETCHER,JR. ROBERT GOTTSCHALK Attesting Officer Commissioner of Patents IORM po'wso $69) USCOMM-DC eo37s-pe9 

